The delivery of materials across the Blood Brain Barrier (BBB) to specific cellular targets is a critical challenge for the study and treatment of diseases and disorders of the brain. New methods for targeted brain delivery will dramatically improve the efficacy of both new and existing therapeutics. We propose to develop a collection of safe, effective, and widely usable peptide-based carriers that are able to deliver exogenous agents across the BBB directly to specific cell populations in the central nervous system (CNS). Recently, Cell Penetrating Peptides (CPPs) have been discovered that are capable of delivering cargos across biological membranes, including the BBB. But their therapeutic applications are limited, as they lack cell and tissue-type specificity. Homing peptides have been identified that target specific cell types, but their application is hampered by an inability to deliver cargos across membranes. In this proposal, we will use Directed Evolution to engineer novel Specific Cell Penetrating Peptides (SCPPs) that traverse the BBB and deliver materials to specific cell types. Our goal will be accomplished using a novel plasmid display system which can be used to efficiently identify SCPPs with specific cell-type penetrating activities. The plasmid display system provides a linkage between the SCPP sequence and the DNA sequence that encodes it. And, the associated plasmid serves as a cargo that can be used as a functional marker to indicate cellular penetration. Large randomized plasmid display libraries will be screened using an in vitro model of the BBB consisting of organotypic brain slice cultures grown on a confluent layer of primary endothelial cells. Fluorescence activated cell sorting (FACS) will be used to isolate specific cell types that were penetrated by the SCPPs, and the SCPPs will be identified through DNA sequencing of the attached plasmid cargo. SCPPs that are able to penetrate the BBB in vitro will be screened in vivo using an anesthetized rat. The in vivo screening will ensure that the SCPPs possess highly specific brain targeting activities and that non- specific SCPPs are eliminated from the library. The novel SCPPs engineered through this Directed Evolution approach should find immediate utility in neuroscience and behavioral research, as well as the treatment of neurodegenerative diseases. The Blood Brain Barrier (BBB) restricts the access of most materials to the central nervous system (CNS). New peptide-based carriers engineered to deliver exogenous agents across the BBB to specific cell-types in the brain will open new avenues of neuroscience and behavioral research, and they will enable new methods for the targeted delivery of therapeutic material for the treatment of various diseases of the brain. [unreadable] [unreadable] [unreadable]